T cells are a part of the adaptive immune system that scan the intracellular environment in order to target and destroy infected cells. Small peptide fragments, representing the entire cellular content, are transported to the cell surface as pMHCs, allowing T cell surface expressed antigen specific T cell receptors to scan for foreign signals. T cells interact with a large number of different cell types and recognize a diverse array of pathogens. There are distinct antigen recognition pathways which generate the appropriate T cell response. T cell activation can lead to a number of immune responses such as antibody production, activation of phagocytic cells and direct cell killing.
T helper cells (Th cells) are a subset of αβT-cells that usually express the CD4 co-receptor and have a major role in controlling and regulating the immune system by affecting other white blood cells. Th cells are vital to human immune responses because they orchestrate the immune system by controlling other T cell subsets, B cells and innate immune responses. The Th cell response is defined by two distinct pathways involving two different subtypes of Th cell, Th1 and Th2 cells. Th1 cells are largely targeted towards intravesicular pathogens such as bacteria and parasites via the activation of infected macrophages. Th2 cells largely invoke antibody production in B cells that neutralize extracellular pathogens and toxins. Th cell activation leads to the induction of a number of pathways that can result in B cell antibody production and immunoglobulin class switching, and macrophage action via both direct interaction and through the release of soluble factors.
Memory T cells are derived from normal T cells that have learned to remember the strategy used to defeat previous infections. Adaptive immunity is said to have memory because the immune system learns giving, for instance, life long immunity to infections such as mumps or chicken pox. T cells constitute a highly evolved arm of the adaptive immune system that is able to distinguish between pathogens and is capable of evolving or adapting during the lifetime of an individual such that immunity becomes better with each successive exposure to pathogen. Following infection, some of the activated T-cells become memory cells that remain in a state of readiness and have the ability to rapidly expand and fight recurrence of the same disease. That is, these cells learn from their experience of fighting a particular infection and so can use the most effective strategy to manage the same infection later.
Th17 cells, the T helper cells that produce IL-17 (also referred to as IL-17A) and other pro-inflammatory cytokines, have been shown to have key functions in a wide variety of autoimmune disease models in mice and are thought to be similarly involved in human disease (Weaver, et al., Annual Review of Immunology 2007; 25: 821-852; Bettelli, et al., Current Opinion in Immunology 2007; 19: 652-657; Stockinger, et al., Current Opinion in Immunology 2007; 19: 281-286). In healthy humans, IL-17-secreting cells are present in the CD45RO+CCR6+ populations of T cells from peripheral blood (Acosta-Rodriguez, et al., Nature Immunology 2007; 8: 639-646; Annunziato, et al., J Exp Med 2007; 204: 1849-1861) and gut (Annunziato, et al., J Exp Med 2007; 204: 1849-1861). Th17 cells or their products have been associated with the pathology of multiple inflammatory or autoimmune disorders in humans. IL-17 protein and Th17 CD4+ T cells are found in lesions from multiple sclerosis patients (Lock, et al., Nat Med 2002; 8, 500-508; Matusevicius, et al., Mult Scler 1999; 5; 101-104; Tzartos, et al., Am J Pathol 2008; 172: 146-155) where they are thought to contribute to the disruption of the blood-brain barrier (Kebir, et al., Nat Med 2007; 13: 1173-1175). IL-17 is produced by CD4+ T cells of rheumatoid synovium (Chabaud, et al., Arthritis Rheum 1999; 42: 963-970) and is thought to contribute to inflammation in rheumatoid arthritis (Attur, et al., Arthritis Rheum 1997; 40: 1050-1053; Fossiez, et al., J Exp Med 1996; 183: 2593-2603). In psoriasis, products associated with Th17 cells, including IL-17, IL-17F, IL-22, and CCR6 are induced (Homey, et al., J Immunol 2000; 164: 6621-6632; Zheng, et al., Nature 2007; 445: 648-651; Wilson, et al., Nature Immunology 2007; 8: 950-957). IL-17 is induced in the gut mucosa from Crohn's disease and ulcerative colitis patients and Th17 cells are detected (Homey, et al., J Immunol 2000; 164: 6621-6632; Annunziato, et al., J Exp Med 2007; 204: 1849-1861). IL-23, which is produced by dendritic cells in the intestine (Becker, et al., The Journal of Clinical Investigation 2003; 112: 693-706), contributes significantly to Th17 cell differentiation (McGeachy, et al., Nature Immunology 2007; 8: 1390-1397). Strikingly, polymorphisms in the IL23R gene are associated with Crohn's disease, further implicating the Th17 cell pathway in the pathogenesis of this disease (Duerr, et al., Science 2006; 314: 1461-1463).
The mechanisms leading to differentiation of Th17 cells have been well established in mice but they are still poorly understood in humans. In mice, differentiation of Th17 cells that secrete IL-17 and IL-17F requires the expression of the transcription factors Rorγt, an orphan nuclear hormone receptor, STAT3 and IRF4 (reviewed in Ivanov, I I, et al., Semin Immunol 2007). Rorγt is sufficient to direct expression of IL-17 in activated mouse T cells (Ivanov, I I, et al. Cell 2006; 126: 1121-1133) and is thus considered to be the effector transcription factor that establishes the Th17 differentiation lineage. Conditions that induce Th17 cell differentiation from naive murine T cells, including expression of Rorγt, have been established. Combinations of TGF-β and IL-6 (Veldhoen, et al., Immunity 2006; 24: 179-189; Bettelli, et al., Nature 2006; 441: 235-238; Mangan, et al., Nature 2006; 441: 231-234) or TGF-β and IL-21 (Korn, et al., Nature 2007; 448: 484-487; Nurieva, et al., Nature 2007; 448: 480-483; Zhou, et al., Nature Immunology 2007; 8: 967-974) are sufficient to initiate IL-17 and IL-17F expression. Expression of IL-22, considered to be another Th17 cytokine, is induced by IL-6 and inhibited by high concentrations of TGF-β (Zheng, et al., Nature 2007; 445: 648-651). IL-23 is required in vivo for the generation of pathogenic Th17 cells, but it is not required in vitro for the induction of IL-17, IL-17F or IL-22 (McGeachy, et al., Nature Immunology 2007; 8: 1390-1397).
In contrast to murine T cells, human T cells with a naive surface phenotype fail to produce IL-17 in the presence of TGF-β and IL-6 (Chen, et al., Arthritis Rheum 2007; 56: 2936-2946; Acosta-Rodriguez, et al., Nature Immunology 2007; 8: 942-949; van Beelen, et al., Immunity 2007; 27: 660-669; Evans, et al., Proc Natl Acad Sci USA 2007; 104: 17034-17039). Increased expression of IL-17 was, however, observed by some groups in response to IL-1 alone (Acosta-Rodriguez, et al., Nature Immunology 2007; 8: 942-949) or with IL-23 (Wilson, et al., Nature Immunology 2007; 8: 950-957). Others have failed to observe such a response (van Beelen, et al., Immunity 2007; 27: 660-669). These disparate findings reveal that the identities of the exogenous factors required to induce the differentiation of human Th17 cells remain unknown. The difference between the requirements for mouse and human Th17 cell differentiation have been ascribed to divergent differentiation processes, although it remains possible that T cells purified from adult peripheral blood on the basis of CD45RA expression alone are not equivalent to naive murine T cells (Stockinger, et al., Semin Immunol 2007; De Rosa, et al., Nat Med 2001; 7: 245-248; Laurence, et al., Nature Immunology 2007; 8: 903-905).
In addition to Th1 and Th2 cells, it is now accepted that naïve CD4+ T cells can differentiate into Th17 or Th22 cells that secrete IL-17 or IL-22, respectively (Annunziato, et al., Arthritis Res Ther 2009; 11: 257; Wolk, et al., Semin Immunopathol 2010; 32: 17-31; Zhou, et al., Immunity 2009; 30: 646-655). Th17 cells mediate pro-inflammatory immune responses against some species of extracellular bacteria and most fungal pathogens (Bettelli, et al., Nature 2006; 441: 235-238; de Beaucoudrey, et al., J Exp Med 2008; 205: 1543-1550; Holland, et al. n Engl J Med 2007; 357: 1608-1619; Minegishi, et al., Nature 2007; 448: 1058-1062). Th17 cells are also broadly implicated in the pathogenesis of many common autoimmune disorders, including multiple sclerosis, rheumatoid arthritis, psoriasis, and inflammatory bowel disease (Cua, et al., Nature 2003; 421: 744-748; Fujino, et al., Gut 2003; 52: 65-70; Langrish, et al., J Exp Med 2005; 201: 233-240; Lock, et al., Nature Medicine 2002; 8: 500-508; Wilson, et al., Nature Immunology 2007; 8: 950-957). Th22 cells are involved in skin immunity and remodeling, but are also implicated in cutaneous inflammatory conditions such as psoriasis (Eyerich, et al., The Journal of Clinical Investigation 2009; 119: 3573-3585; Wolk, et al., Semin Immunopathol 2010; 32: 17-31). Ex vivo analyses of human peripheral blood T cells indicate that nearly all IL-17-secreting cells, and the majority of IL-22-producing T cells, express the chemokine receptor CCR6 and the transcription factor RORC (Annunziato, et al., J Exp Med 2007; 204: 1849-1861; Duhen, et al., Nature Immunology 2009; 10: 857-863; El Hed, et al., J Infect Dis 2010; 201: 843-854; Romagnani, et al., Mol Immunol 2009; 47: 3-7; Singh, et al., J Immunol 2008; 180: 214-221). Accordingly, ectopic expression of RORC in naïve T cells or CCR6− memory cells is sufficient to induce IL-17 secretion (Manel, et al., Nature Immunology 2008; 9: 641-649). Currently, all effector T cell subsets, including Th17 and Th22 cells, are defined solely based on cytokine expression following ex vivo stimulation (Duhen, et al., Nature Immunology 2009). However, it is not clear whether this definition accurately reflects the full repertoire of memory T cells that have the capacity to produce IL-17/IL-22 at sites of inflammation. Moreover, the cytokines and downstream signaling pathways that control IL-17/IL-22 secretion in lineage-committed memory T cells remain uncharacterized.
The IL-2 family of cytokines, which signal through multimeric receptors containing the shared common gamma chain (γc) subunit, includes IL-2, IL-4, IL-7 IL-9, IL-15, and IL-21. These cytokines, particularly IL-2, IL-7, and IL-15, play pivotal roles in promoting T cell development, homeostasis, and differentiation. In addition to activating Jak/Stat pathways, γc-signaling induces the generation of lipid second messengers through activation of PI-3K (Rochman, et al., Nature Reviews 2009; 9: 480-490). One of these second messengers, phosphatidylinositol-(3,4,5)-trisphosphate (PI(3,4,5)P3), binds to the pleckstrin homology (PH) domain of proteins and controls the activity and function of a number of signaling molecules, including the serine/threonine protein kinase Akt (Fruman, Current Opinion in Immunology 2004; 16: 314-320). In turn, Akt directly phosphorylates the transcription factor Forkhead box protein O1 (FOXO1), thereby preventing its nuclear translocation and transcriptional activity (Brunet, et al., Cell 1999; 96: 857-868) Inhibition of FOXO1 by PI-3K has been shown to be essential for γc-cytokine signaling-mediated cell survival, proliferation and glucose utilization in leukemia cells (Barata, et al., J Exp Med 2004; 200: 659-669). In contrast, activation of FOXO1, by way of reduced PI-3K signaling, can lead to the expression of another transcription factor called kruppel-like factor 2 (KLF2) (Kerdiles, et al., Nature Immunology 2009; 10: 176-184; Sinclair, et al., Nature Immunology 2008; 9: 513-521), which has been implicated in the modulation of IFNγ and IL-4 production in human and mouse T cells (Bu, et al., J Clin Invest 2010; 120: 1961-1970; Weinreich, et al., Nature Immunology 2010; 11: 709-716; Weinreich, et al., Immunity 2009; 31: 122-130). However, whether FOXO1 or KLF2 act downstream of PI-3K to regulate effector cytokine production in human memory T cells is not yet known.
Common gamma chain utilizing cytokines, γc-cytokines, notably IL-2, IL-7 or IL-15, are sufficient to induce de novo expression of IL-17, IL-22, and other Th17-signature cytokines in CCR6+, but not CCR6−, TM cells. Treatment of cytokine-stimulated CCR6+ TM cells with small molecule inhibitors of PI-3K or Akt repressed γc-cytokine-driven IL-17/IL-22 expression, as did ectopic expression of FOXO1 or KLF2. These findings suggest that PI-3K signaling may amplify Th17-/Th22-associated tissue inflammation by promoting pro-inflammatory cytokine expression in lineage-committed human Th17/Th22 cells. Our results also demonstrate that the frequency of human memory T cells harboring the capacity to secrete IL-17/IL-22 in vivo may be substantially higher than what is predicted based on ex vivo cytokine analysis.
Phosphatidylinositol 3-kinases (PI 3-kinases or PI-3Ks) are a family of enzymes involved in cellular functions such as cell growth, proliferation, differentiation, motility, survival and intracellular trafficking, which in turn are involved in cancer. PI-3Ks are a family of related intracellular signal transducer enzymes capable of phosphorylating the 3 position hydroxyl group of the inositol ring of phosphatidylinositol (PtdIns). The pathway, with oncogene PIK3CA and tumor suppressor PTEN (gene), is implicated in insensitivity of cancer tumors to insulin and IGF1, in calorie restriction.
PI-3Ks interact with the IRS (Insulin receptor substrate) in order to regulate glucose uptake through a series of phosphorylation events. Discrete members of PI-3K family are activated in immune system depending on the type of cell and/or receptor. (Koyasu, Nature Review, 2003; the role of PI3K in immune system). The phosphoinositol-3-kinase family is divided into three different classes: Class I, Class II, and Class III. The classifications are based on primary structure, regulation, and in vitro lipid substrate specificity.
Class I PI-3Ks are responsible for the production of Phosphatidylinositol 3-phosphate (PI(3)P), Phosphatidylinositol (3,4)-bisphosphate (PI(3,4)P2), and Phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3. The PI-3K is activated by G protein-coupled receptors and tyrosine kinase receptors. Class I PI-3K are heterodimeric molecules composed of a regulatory and a catalytic subunit; they are further divided between IA and IB subsets based on sequence similarity. Class IA PI-3K is composed of a heterodimer between a p110 catalytic subunit and a p85 regulatory subunit. There are five variants of the p85 regulatory subunit, designated p85α, p55α, p50α, p85β, or p55γ. There are also three variants of the p110 catalytic subunit designated p110α, β, or δ. The first three regulatory subunits are all splice variants of the same gene (Pik3r1), the other two being expressed by other genes (Pik3r2 and Pik3r3, p85β, and p55γ, respectively). The most highly expressed regulatory subunit is p85α; all three catalytic subunits are expressed by separate genes (Pik3ca, Pik3cb, and Pik3cd for p110α, p110β, and p110δ, respectively). The first two p110 isoforms (α and β) are expressed in all cells, but p110δ is expressed primarily in leukocytes, and it has been suggested that it evolved in parallel with the adaptive immune system. The regulatory p101 and catalytic p110γ subunits comprise the type IB PI-3K and are encoded by a single gene each.
The majority of the research on PI 3-kinases has focused on the Class I PI 3-kinases. Class I PI 3-kinases are composed of a catalytic subunit known as p110 and a regulatory subunit related to either p85 or p101. The p85 subunits contain SH2 and SH3 domains (Online ‘Mendelian Inheritance in Man’ (OMIM) 171833). The SH2 domains bind preferentially to phosphorylated tyrosine residues in the amino acid sequence context Y-X-X-M.
Class II and III PI-3K are differentiated from the Class I by their structure and function. Class II comprises three catalytic isoforms (C2α, C2β, and C2γ), but, unlike Classes I and III, no regulatory proteins. Class II catalyse the production of PI(3)P and PI(3,4)P2 from PI; however, little is known about their role in immune cells. C2α and C2β are expressed through the body, however expression of C2γ is limited to hepatocytes. The distinct feature of Class II PI-3Ks is the C-terminal C2 domain. This domain lacks critical Asp residues to coordinate binding of Ca2+, which suggests class II PI-3Ks bind lipids in a Ca2+-independent manner.
Class III produces only PI(3)P from PI but are more similar to Class I in structure, as they exist as a heterodimers of a catalytic (Vps34) and a regulatory (Vps15/p150) subunits. Class III seems to be primarily involved in the trafficking of proteins and vesicles. There is, however, evidence to show that they are able to contribute to the effectiveness of several process important to immune cells, not least phagocytosis.
The various 3-phosphorylated phosphoinositides that are produced by PI 3-kinases (PtdIns3P, PtdIns(3,4)P2, PtdIns(3,5)P2, and PtdIns(3,4,5)P3) function in a mechanism by which an assorted group of signalling proteins, containing PX domain, pleckstrin homology domains (PH domains), FYVE domains and other phosphoinositide-binding domains, are recruited to various cellular membranes.
PI 3-kinases have been linked to an extraordinarily diverse group of cellular functions, including cell growth, proliferation, differentiation, motility, survival and intracellular trafficking Many of these functions relate to the ability of class I PI 3-kinases to activate protein kinase B (PKB, aka Akt) as in the PI-3K/AKT/mTOR pathway. The p110δ and p110γ isoforms regulate different aspects of immune responses. PI 3-kinases are also a key component of the insulin signaling pathway. Hence there is great interest in the role of PI 3-kinase signaling in Diabetes mellitus.
The pleckstrin homology domain of AKT binds directly to PtdIns(3,4,5)P3 and PtdIns(3,4)P2, which are produced by activated PI 3-kinase.ince PtdIns(3,4,5)P3 and PtdIns(3,4)P2 are restricted to the plasma membrane, this results in translocation of AKT to the plasma membrane. Likewise, the phosphoinositide-dependent protein kinase 1 (PDK1 or, rarely referred to as PDPK1) also contains a pleckstrin homology domain that binds directly to PtdIns(3,4,5)P3 and PtdIns(3,4)P2, causing it to also translocate to the plasma membrane upon activation of PI 3-kinase. The colocalization of activated PDK1 and AKT allows AKT to become phosphorylated by PDK1 on threonine 308, leading to partial activation of AKT. Full activation of AKT occurs upon phosphorylation of serine 473 by the TORC2 complex of the mTOR protein kinase. (The nomenclature can be confusing. Note that PDK1 also refers to the unrelated enzyme Pyruvate dehydrogenase kinase, isozyme 1. Similarly, TORC2 also refers to the unrelated transcription factor Transducer of Regulated CREB activity 2, which has recently been renamed CREB-regulated transcription coactivator 2 (CRTC2) to reduce the confusion). The “PI3-k/AKT” signaling pathway has been shown to be required for an extremely diverse array of cellular activities—most notably cellular proliferation and survival.
Many other proteins have been identified that are regulated by PtdIns(3,4,5)P3, including Bruton's Tyrosine Kinase (BTK), General Receptor for Phosphoinositides-1 (GRP1), and the O-linked N-acetylglucosamine (O-GlcNAc) transferase.
The class IA PI 3-kinase p110α is mutated in many cancers. Many of these mutations cause the kinase to be more active. The PtdIns(3,4,5)P3 phosphatase PTEN that antagonises PI 3-kinase signaling is absent from many tumours. Hence, PI 3-kinase activity contributes significantly to cellular transformation and the development of cancer.
PI-3K has also been implicated in Long-term potentiation (LTP). Whether it is required for the expression or the induction of LTP is still debated. In mouse hippocampal CA1 neurons, PI-3K is complexed with AMPA Receptors and compartmentalized at the postsynaptic density of glutamatergic synapses. PI-3K is phosphorylated upon NMDA Receptor-dependent CaMKII activity, and it then facilitates the insertion of AMPA-R GluR1 subunits into the plasma membrane. This suggests that PI-3K is required for the expression of LTP. Furthermore, PI-3K inhibitors abolished the expression of LTP in rat hippocampal CA1, but do not affect its induction. Notably, the dependence of late-phase LTP expression on PI-3K seems to decrease over time.
However, another study found that PI-3K inhibitors suppressed the induction, but not the expression, of LTP in mouse hippocampal CA1. The PI-3K pathway also recruits many other proteins downstream, including mTOR, GSK3β, and PSD-95. The PI-3K-mTOR pathway leads to the phosphorylation of p70S6K, a kinase that facilitates translational activity further suggesting that PI-3K is required for the protein-synthesis phase of LTP induction instead.
Many of the PI 3-kinases appear to have a serine/threonine kinase activity in vitro; however, it is unclear whether this has any role in vivo.
In addition to the class I-class III PI 3-kinases there is a group of more distantly related enzymes that are sometimes referred to as class IV PI 3-kinases. The class IV PI 3-kinases family is composed of ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3 related (ATR), DNA-dependent protein kinase (DNA-PK) and mammalian Target Of Rapamycin (mTOR). These members of the PI 3-kinase superfamily are protein serine/threonine kinases.
All PI 3-kinases are inhibited by the drugs wortmannin and LY294002, although certain members of the class II PI 3-kinase family show decreased sensitivity. As wortmannin and LY294002 are broad inhibitors against PI 3-kinases and a number of unrelated proteins at higher concentrations they are too toxic to be used as therapeutics. A number of pharmaceutical companies have recently been working on PI 3-kinase isoform specific inhibitors including the class I PI 3-kinase, p110δ isoform specific inhibitors, IC486068 and IC87114, ICOS Corporation.
FOX (Forkhead box) proteins are a family of transcription factors that play important roles in regulating the expression of genes involved in cell growth, proliferation, differentiation, and longevity. Many FOX proteins are important to embryonic development. The defining feature of FOX proteins is the forkhead box, a sequence of 80 to 100 amino acids forming a motif that binds to DNA. This forkhead motif is also known as the winged helix due to the butterfly-like appearance of the loops in the protein structure of the domain. Forkhead genes are a subgroup of the helix-turn-helix class of proteins. Many other genes encoding FOX proteins have been identified. For example, the FOXF2 gene encodes forkhead box F2, one of many human homologues of the Drosophila melanogaster transcription factor forkhead. FOXF2 is expressed in lung and placenta. Some FOX genes are downstream targets of the hedgehog signaling pathway, which plays a role in the development of basal cell carcinomas. Members of the class O regulate metabolism, cellular proliferation, stress tolerance and possibly lifespan. The activity of FoxO is controlled by post-translational modifications, including phosphorylation, acetylation and ubiquitination.
The Sp/KLF family (specificity protein/Krüppel-like factor) is a family of transcription factors, including the Kruppel-like factors as well as Sp1, Sp2, Sp3, Sp4, Sp8, Sp9; and possibly Sp5 and Sp7. KLF14 is also designated Sp6. The Krüppel-like family of transcription factors (Klfs), have been extensively studied for their roles in cell proliferation, differentiation and survival, especially in the context of cancer. All KLF family members are characterized by their three Cys2 His2 zinc fingers located at the C-terminus separated by a highly conserved H/C link. DNA binding studies demonstrated that the KLFs have similar affinities for different GC-rich sites, or sites with CACCC homology, and can compete with each other for the occupation of such sites. KLFs also share a high degree of homology between the specificity protein (Sp) family of zinc-finger transcription factors and bind similar, if not the same sites, in a large number of genes. The following human genes encode Kruppel-like factors: KLF1, KLF2, KLF3, KLF4, KLF5, KLF6, KLF7, KLF8, KLF9, KLF10, KLF11, KLF12, KLF13, KLF14, KLF15, KLF16, and KLF17.